ABSTRACT. Monoclonal antibodies are used for treatment of a wide range of diseases from cancer to infectious diseases. However, development of antibodies with high affinity and specificity is still time- consuming, labor-intensive and unpredictable, especially to low immunogenic or toxic targets. A new approach for rapid development of antibodies against a variety of diseases, especially against emerging diseases is much needed. Abzyme Therapeutics LLC is actively developing animal-free antibody discovery platforms to accelerate generation of diagnostic and therapeutic antibodies. Phase I objectives have been successfully completed. Specifically, we have created a so-called yeast surface display camelid Self-Diversifying VHH Antibody Library or SDALib as a monoclonal antibody generating system providing a diverse array of antibodies in vitro, without using in vivo immunization. The main feature of the system is the ability to self-generate a library with diversified antibodies by means of hypermutation induction in antibody encoding genes. As proof-of-principle, we have successfully used the platform to isolate a set of single-domain antibodies against N1 neuraminidase (NA) derived from influenza H5N1 virus. Obtained VHH antibodies have been shown to express efficiently in a bacterial system providing an opportunity for accelerated scale-up of antibody production for therapeutic application. Anti-NA VHH antibodies have been purified and characterized in in vitro assays. As proof of principle one (Flu27-8) out of several anti-NA VHH antibodies, that is reactive with N1 NAs of both H5N1 and H1N1 influenza viruses, has been humanized preserving the full anti-NA activity of the parental molecule. Bi- valent anti-NA antibodies that have been produced in a bacterial system as linear fusions showed a significant increase in antibody activity in in vitro assays. Subsequent efforts of Phase II consist of three specific aims that will be performed simultaneously: (i) advance the N1 anti-NA antibodies isolated in Phase I toward clinical application by demonstrating both in vitro and in vivo activity against H5N1 and H1N1 influenza viruses; (ii) expand the application of the platform for developing antibodies targeting both hemagglutinin and NAs of other influenza virus subtypes, e.g. H3N2 and H7N9 which have unusual zoonotic potential and aiming for antibodies cross-reacting with several different neuraminidases (H5N1, H1N1, H3N2 and H7N9); (iii) utilize the antibody discovery platform for developing VHH antibodies against a set of cancer targets followed by production of novel IgG-like VHH-based bispecific humanized antibodies. Phase II efforts are to further extend the application of the technology for rapid development of novel antibodies to therapeutic targets and to advance humanized anti-influenza antibodies isolated in phase I into clinical studies.